U.S. patent application number 13/518534 was filed with the patent office on 2013-01-31 for methods for producing isomers of muconic acid and muconate salts.
This patent application is currently assigned to Amyris, Inc.. The applicant listed for this patent is Vu Bui, Man Kit Lau, Doug MacRae, Dirk Schweitzer. Invention is credited to Vu Bui, Man Kit Lau, Doug MacRae, Dirk Schweitzer.
Application Number | 20130030215 13/518534 |
Document ID | / |
Family ID | 43585641 |
Filed Date | 2013-01-31 |
United States Patent
Application |
20130030215 |
Kind Code |
A1 |
Bui; Vu ; et al. |
January 31, 2013 |
Methods for Producing Isomers of Muconic Acid and Muconate
Salts
Abstract
A method for producing cis,trans- and trans,trans-isomers of
muconate by providing cis,cis-muconate produced from a renewable
carbon source through biocatalytic conversion; isomerizing
cis,cis-muconate to cis,trans-muconate under reaction conditions in
which substantially all of the cis,cis-muconate is isomerized to
cis,trans-muconate; separating the cis,trans-muconate; and
crystallizing the cis,trans-muconate. The cis,trans-isomer can be
further isomerized to the trans,trans-isomer. In one example, the
method includes culturing recombinant cells that express
3-dehydroshikimate dehydratase, protocatechuate decarboxylase and
catechol 1,2-dioxygenase in a medium comprising the renewable
carbon source and under conditions in which the renewable carbon
source is converted to 3-dehydroshikimate by enzymes in the common
pathway of aromatic amino acid biosynthesis of the cell, and the
3-dehydroshikimate is biocatalytically converted to
cis,cis-muconate.
Inventors: |
Bui; Vu; (Davis, CA)
; Lau; Man Kit; (Minnaepolis, MN) ; MacRae;
Doug; (Okemos, MI) ; Schweitzer; Dirk;
(Okemos, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bui; Vu
Lau; Man Kit
MacRae; Doug
Schweitzer; Dirk |
Davis
Minnaepolis
Okemos
Okemos |
CA
MN
MI
MI |
US
US
US
US |
|
|
Assignee: |
Amyris, Inc.
Emeryville
CA
|
Family ID: |
43585641 |
Appl. No.: |
13/518534 |
Filed: |
January 10, 2011 |
PCT Filed: |
January 10, 2011 |
PCT NO: |
PCT/US11/20681 |
371 Date: |
October 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61335638 |
Jan 8, 2010 |
|
|
|
Current U.S.
Class: |
562/591 ;
435/142 |
Current CPC
Class: |
C12N 9/88 20130101; C12P
7/44 20130101; C12N 9/0069 20130101 |
Class at
Publication: |
562/591 ;
435/142 |
International
Class: |
C12P 7/44 20060101
C12P007/44; C07C 51/353 20060101 C07C051/353 |
Claims
1. A method for producing cis,trans-muconate comprising: providing
cis,cis-muconate produced from a renewable carbon source through
biocatalytic conversion; isomerizing cis,cis-muconate to
cis,trans-muconate under reaction conditions in which substantially
all of the cis,cis-muconate is isomerized to cis,trans-muconate;
separating the cis,trans-muconate; and crystallizing the
cis,trans-muconate.
2. The method of claim 1, further comprising culturing recombinant
cells that express 3-dehydroshikimate dehydratase, protocatechuate
decarboxylase and catechol 1,2-dioxygenase in a medium comprising
the renewable carbon source and under conditions in which the
renewable carbon source is converted to 3-dehydroshikimate by
enzymes in the common pathway of aromatic amino acid biosynthesis
of the cell, and the 3-dehydroshikimate is biocatalytically
converted to cis,cis-muconate.
3. The method of claim 1, wherein the isomerization reaction is
catalyzed by an acid, wherein the acid can be an inorganic acid or
an organic acid.
4-8. (canceled)
9. The method of claim 1, wherein the separating step comprises
precipitating the cis,trans-muconate from solution by
acidification.
10-11. (canceled)
12. The method of claim 1, wherein the cis,trans-muconate is
crystallized with an organic solvent, wherein the organic solvent
comprises one or more of methanol, ethanol, propanol, isopropanol,
butanol, acetic acid, acetonitrile, acetone, and
tetrahydrofuran.
13-16. (canceled)
17. The method of claim 1, wherein the cis,trans-muconate is
isomerized to trans,trans-muconate in a reaction catalyzed by
I.sub.2, by a precious metal hydrogenation catalyst, by a sponge
metal hydrogenation catalyst, or by a skeletal hydrogenation
catalyst.
18. A method for producing cis,trans-muconate comprising: providing
a fermentation broth comprising cis,cis-muconate produced from a
renewable carbon source through biocatalytic conversion;
isomerizing cis,cis-muconate to cis,trans-muconate under reaction
conditions in which substantially all of the cis,cis-muconate is
isomerized to cis,trans-muconate; separating the cis,trans-muconate
from the broth; and crystallizing the cis,trans-muconate.
19. The method of claim 18, wherein the fermentation broth
comprises recombinant cells that express 3-dehydroshikimate
dehydratase, protocatechuate decarboxylase and catechol
1,2-dioxygenase.
20-21. (canceled)
22. The method of claim 19, further comprising: culturing the
recombinant cells that express 3-dehydroshikimate dehydratase,
protocatechuate decarboxylase and catechol 1,2-dioxygenase in a
medium comprising the renewable carbon source and under conditions
in which the renewable carbon source is converted to
3-dehydroshikimate by enzymes in the common pathway of aromatic
amino acid biosynthesis of the cell, and the 3-dehydroshikimate is
biocatalytically converted to cis,cis-muconate, and wherein the
recombinant cells are cultured in a fermentor vessel, thereby
producing the fermentation broth.
23-27. (canceled)
28. The method of claim 1, wherein providing cis,cis-muconate
produced from the renewable carbon source through biocatalytic
conversion employs a host cell transformed with heterologous
structural genes from Klebsiella pneumoniae, which express the
enzymes 3-dehydroshikimate dehydratase and protocatechuate
decarboxylase, and from Acinetobacter calcoaceticus, which
expresses the enzyme catechol 1,2-dioxygenase.
29. The method of claim 28 wherein the host cell further comprises
heterologous DNA sequences which express the enzymes
3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and
3-dehydroquinate synthase.
30. The method of claim 29 wherein the host cell further comprises
heterologous DNA sequences which express the enzyme
transketolase.
31. (canceled)
32. The method of claim 28, wherein the host cell is selected from
mutant cell lines having mutations in the common pathway of
aromatic amino acid biosynthesis that block conversion of
3-dehydroshikimate to chorismate.
33. The method of claim 28, wherein the host cell produces
cis,cis-muconic acid at a rate of at least about 0.95
millimoles/liter/hour.
34. The method of claim 1, wherein providing cis,cis-muconate
produced from the renewable carbon source through biocatalytic
conversion comprises culturing a transformed host cell, which
expresses heterologous structural genes encoding 3-dehydroshikimate
dehydratase, protocatechuate decarboxylase, catechol
1,2-dioxygenase, transketolase, 3-deoxy-D-arabino-heptulosonate
7-phosphate synthase, and 3-dehydroquinate synthase, in a medium
containing a carbon source which is converted to
3-dehydroshikimate, by the enzymes in the common pathway of
aromatic amino acid biosynthesis of the host cell.
35-36. (canceled)
37. A method for producing trans,trans-muconate comprising:
isomerizing one or both of cis,cis-muconate and cis,trans-muconate
produced from a renewable carbon source through biocatalytic
conversion to trans,trans-muconate under reaction conditions in
which substantially all of the cis,cis-muconate or
cis,trans-muconate is isomerized to trans,trans-muconate.
38. The method of claim 37, wherein the isomerization reaction is
catalyzed by a precious metal hydrogenation catalyst, by a sponge
metal hydrogenation catalyst, or by a skeletal hydrogenation
catalyst.
39-41. (canceled)
42. The method of claim 1, wherein the renewable carbon source is a
biospheric feedstock.
43. The method of claim 2, wherein the recombinant cells are
recombinant prokaryotic or eukaryotic cells.
44. The method of claim 18, wherein the renewable carbon source is
a biospheric feedstock.
45. The method of claim 19, wherein the recombinant cells are
recombinant prokaryotic or eukaryotic cells.
46. The method of claim 37, wherein the renewable carbon source is
a biospheric feedstock.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Patent Application No. 61/335,638, filed Jan. 8, 2010,
the disclosures of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to the biological production
of muconate from renewable feedstock. The invention relates more
particularly to the production of muconate isomers, as well as
precursors and derivatives thereof, from a renewable
biomass-derived carbon source.
BACKGROUND OF THE INVENTION
[0003] Worldwide consumption of dimethyl terephthalate (DMT) is
projected to average to 3.97 million metric tons by 2012. DMT is an
ester of terephthalic acid and methanol and is used in the
production of polyesters, including polyethylene terephthalate and
polytrimethylene terephthalate. DMT is also a primary ingredient
used in the manufacture industrial plastics, automotive parts,
films, fishing lines, and food packaging materials.
[0004] Traditionally, DMT production utilizes the esterification of
terephthalic acid with methanol generated by catalytic homogeneous
oxidation of paraxylene. For example, liquid paraxylene can be
oxidized by air in the presence of a cobalt salt catalyst to form
an oxidate containing p-toluic acid and monomethyl terephthalate,
and esterification can be carried out in the presence of methanol
to form DMT.
[0005] Trimellitic acid (TMA) is another commercially important
product with applications as an intermediate in the chemical
industry, including resins for powder coatings, inks, wire enamels,
high performance plasticizers with low volatility, and engineering
polymers for high temperature applications. TMA can also be
dehydrated to produce trimellitic anhydride, which is another
commercially important starting material for the production of
polymers and chemical intermediates.
[0006] Traditionally, TMA is produced by oxidation of pseudocumene
(1,2,4-trimethylbenzene). Terephthalic acid and isophthalic acid
can be produced commercially by liquid phase oxidation of p-xylene
or m-xylene in the presence of acetic acid as a solvent and of a
catalytic system including cobalt, manganese and bromine.
[0007] These processes, like the processes for producing many other
commercially important chemical precursors, intermediates, and
products, can be undesirable due to a heavy reliance upon
environmentally sensitive and non-renewable feedstocks (e.g.,
petroleum feedstocks), and their propensity to yield undesirable
by-products (e.g., greenhouse gases, heavy metals, halogens,
carcinogenic hydrocarbons). As such, a need exists for improved
methods and systems that utilize renewable feedstocks to produce
DMT, TMA, as well as other chemical products.
[0008] As described in U.S. Publication No. 2010/0314243 by Frost
et al. and International Publication No. 2010/148049 by Frost et
al., the disclosures of both of which applications are incorporated
herein by reference in their entirety, DMT and TMA can be produced
from muconic acid. In addition, muconic acid, also known as
2,4-hexadienedioic acid, due to its double bonds and diacid
functionality, can undergo a wide variety of reactions. Many
muconic acid derivatives are known, including lactones, sulfones,
polyamides, polyesters, thioesters, addition polymers, and other
compounds. Such compounds have a wide variety of uses, including
use as surfactants, flame retardants, UV light stabilizers,
thermoset plastics, thermoplastics and coatings. Thus, improved
methods for biological production of muconic acid or muconate from
renewable feedstock are highly desirable for producing DMT, TMA and
other chemicals.
SUMMARY OF THE INVENTION
[0009] The description of the invention uses the terms "muconate"
and "muconic acid." The term "muconic acid" refers to the chemical
species in which both carboxylic acid function groups are
protonated, and the molecule is formally a neutral species. Muconic
acid has the chemical formula HOOC--CH.dbd.CH--CH.dbd.CH--COOH. The
term "muconate" refers to the corresponding deprotonated chemical
species in which one or both of the carboxylic acid function groups
is deprotonated to give the anionic or doubly-anionic form which
would be the predominate chemical species at physiological pH
values. However, as the terms "muconic acid" and "muconate" refer
to the protonated or deprotonated forms of the same molecule, the
terms are used synonymously where the difference between protonated
and deprotonated (e.g., non-ionized and ionized) forms of the
molecule is not usefully distinguished.
[0010] The present invention provides methods for the production
the three isomers of muconate, that is, the cis,cis; cis,trans; and
trans,trans isomers as well as precursors and derivatives thereof,
from biomass-derived carbon sources. The isomers structurally
differ by the geometry around the two double bonds. In addition,
the isomers can have different physical properties (e.g., melting
point) and chemical reactivities. The methods can include microbial
biosynthesis of products from readily available carbon sources
capable of biocatalytic conversion to erythrose 4-phosphate (E4P)
and phosphoenolpyruvate (PEP) in microorganisms having a common
pathway of aromatic amino acid biosynthesis.
[0011] One preferred carbon source is D-glucose. Advantageously,
D-glucose and other carbon sources useable in connection with the
present invention are non-toxic. Furthermore, such carbon sources
are renewable, being derived from starch, cellulose, and sugars
found in corn, sugar cane, sugar beets, wood pulp, and other
biomass resources.
[0012] Host microbial organisms suitable for facilitating various
steps in the present invention can be selected from genera
possessing an endogenous common pathway of aromatic amino acid
biosynthesis. Preferred host organisms include mutant strains of
Escherichia coli genetically engineered to express selected genes
endogenous to Klebsiella pneumoniae and Acinetobacter
calcoaceticus. One preferred E. coli mutant for use in this
invention is E. coli AB2834, an auxotrophic mutant which is unable
to catalyze the conversion of 3-dehydroshikimate (DHS), an
intermediate along the common pathway of aromatic amino acid
biosynthesis, into shikimic acid due to a mutation in the aroE
locus which encodes the enzyme shikimate dehydrogenase.
[0013] The common pathway of aromatic amino acid biosynthesis
produces the aromatic amino acids phenylalanine, tyrosine, and
tryptophan in bacteria and plants. The common pathway ends with the
molecule chorismate, which is subsequently converted into
phenylalanine, tyrosine, and tryptophan by three separate terminal
pathways.
[0014] Approaches for increasing the efficiency of production of
the common aromatic amino acid biosynthetic pathway include those
described in U.S. Pat. No. 5,168,056, issued Dec. 1, 1992, in U.S.
Pat. No. 5,616,496 issued Apr. 1, 1997, and in U.S. Ser. No.
07/994,194, filed Dec. 21, 1992 and now abandoned, the disclosures
of all of which are hereby incorporated by reference in their
entirety.
[0015] In using the genetically engineered host organisms, carbon
flow directed into aromatic amino acid biosynthesis can proceed
along the common pathway to yield elevated intracellular levels of
DHS, which accumulates due to a mutation along the common pathway
of aromatic amino acid biosynthesis, which prevents the conversion
of DHS to chorismate. The DHS serves as a substrate for the enzyme
3-dehydroshikimate dehydratase (aroZ), and action of this enzyme on
DHS produces protocatechuate. Protocatechuate is thereafter
converted to catechol via another enzyme known as protocatechuate
decarboxylase (aroY). The catechol thus formed is in turn converted
to cis,cis-muconic acid by the action of the enzyme catechol
1,2-dioxygenase (catA).
[0016] The three enzymes catalyzing the biosynthesis of
cis,cis-muconate from DHS, that is, aroZ, aroY, and catA, can be
expressed in a host cell using recombinant DNA comprising genes
encoding these three enzymes under control of a suitable promoter.
Carbon flow can be thereby forced away from the pathway of aromatic
amino acid biosynthesis and into the divergent pathway to produce
cis,cis-muconate. The cis,cis-muconic acid thus formed can
accumulate in the extracellular medium which can be separated from
the cells by centrifugation, filtration, or other methods known in
the art. The isolated cis,cis-muconic acid can thereafter be
chemically hydrogenated to yield adipic acid.
[0017] In various embodiments of the invention, after the
cis,cis-muconate has been produced, it can subsequently be
isomerized to cis,trans-muconate or trans,trans-muconate, both of
which have differing physical properties and chemical reactivity
which can give utility different from or beyond that of
cis,cis-muconate. For example, cis,trans-isomer can have greater
solubility than cis,cis-muconate in aqueous and/or organic media,
allowing advantageous recovery and processing. As a further
example, the trans,trans-isomer can have unique utility over the
cis,cis-isomer as a reactant in Diels-Alder reactions.
[0018] In one aspect, the invention features a method for producing
cis,trans-muconate. The method comprises providing cis,cis-muconate
produced from a renewable carbon source through biocatalytic
conversion (e.g., utilizing the aroZ, aroY, and catA enzymes),
isomerizing cis,cis-muconate to cis,trans-muconate under reaction
conditions in which substantially all of the cis,cis-muconate is
isomerized to cis,trans-muconate, separating the
cis,trans-muconate, and crystallizing the separated
cis,trans-muconate (e.g., as the protonated cis,trans-muconic
acid).
[0019] In another aspect, the invention features a method for
producing cis,trans-muconate. The method comprises: providing a
fermentation broth comprising cis,cis-muconate produced from a
renewable carbon source through biocatalytic conversion;
isomerizing cis,cis-muconate to cis,trans-muconate under reaction
conditions in which substantially all of the cis,cis-muconate is
isomerized to cis,trans-muconate; separating the cis,trans-muconate
from the broth; and crystallizing the cis,trans-muconate.
[0020] In yet another aspect, the invention features
cis,trans-muconate produced by a method featured by the invention.
The cis,trans-muconate can be recovered as a salt, for example, an
inorganic salt such as sodium, calcium, or ammonium muconate.
[0021] In yet another aspect, the invention features a method for
producing trans,trans-muconate that includes isomerizing
cis,cis-muconate produced from a renewable carbon source through
biocatalytic conversion to trans,trans-muconate under reaction
conditions in which substantially all of the cis,cis-muconate is
isomerized to trans,trans-muconate. For example, the isomerization
reaction can be catalyzed by a precious metal hydrogenation
catalyst, by a sponge metal hydrogenation catalyst, or by a
skeletal hydrogenation catalyst.
[0022] In yet another aspect, the invention features a method for
producing trans,trans-muconate that includes isomerizing
cis,trans-muconate produced from a renewable carbon source through
biocatalytic conversion to trans,trans-muconate under reaction
conditions in which substantially all of the cis,trans-muconate is
isomerized to trans,trans-muconate. For example, the isomerization
reaction can be catalyzed by a precious metal hydrogenation
catalyst, by a sponge metal hydrogenation catalyst, or by a
skeletal hydrogenation catalyst.
[0023] In still another aspect, the invention features
trans,trans-muconate produced by a method featured by the invention
(e.g., renewable trans,trans-muconate).
[0024] In other examples, any of the aspects above, or any method,
apparatus, or composition of matter described herein, can include
one or more of the following features.
[0025] In various embodiments, the method includes culturing
recombinant cells that express 3-dehydroshikimate dehydratase
(e.g., aroZ), protocatechuate decarboxylase (e.g., aroY) and
catechol 1,2-dioxygenase (e.g., catA) in a medium comprising a
renewable carbon source and under conditions in which such
renewable carbon source is converted to DHS by enzymes found in the
common pathway of aromatic amino acid biosynthesis of the cell, and
the resulting DHS is biocatalytically converted to
cis,cis-muconate.
[0026] The production of cis,cis-muconate by the fermentation of
the renewable carbon source can produce a broth comprising the
recombinant cells and extracellular cis,cis-muconate. The
production can also include the step of separating the recombinant
cells, cell debris, insoluble proteins and other undesired solids
from the broth to give a clarified fermentation broth containing
substantially all, or most of, the cis,cis-muconate formed by the
fermentation. The cis,cis-muconate can then be isomerized to
cis,trans-muconate in the clarified fermentation broth.
[0027] In certain embodiments, a fermentation broth comprising
cis,cis-muconate produced from a renewable carbon source through
biocatalytic conversion can be provided for producing
cis,trans-muconate or trans,trans-muconate. The fermentation broth
can include recombinant cells that express 3-dehydroshikimate
dehydratase, protocatechuate decarboxylase and catechol
1,2-dioxygenase. In some embodiments, the fermentation broth is
provided in a vessel and the isomerization reaction is carried out
in the vessel. The vessel can be a fermentor vessel. In some
examples, recombinant cells that express 3-dehydroshikimate
dehydratase, protocatechuate decarboxylase and catechol
1,2-dioxygenase can be cultured in a medium comprising the
renewable carbon source and under conditions in which the renewable
carbon source is converted to 3-dehydroshikimate by enzymes in the
common pathway of aromatic amino acid biosynthesis of the cell, and
the 3-dehydroshikimate is biocatalytically converted to
cis,cis-muconate. For example, the recombinant cells can be
cultured in the fermentor vessel, thereby producing the
fermentation broth. Additionally, the recombinant cells can be
removed from the fermentation broth as desired.
[0028] In some embodiments, the isomerization reaction is catalyzed
by an acid. The acid can be an inorganic acid (e.g., mineral acid)
or an organic acid. An acid can be applied to the process in either
a hydrated or anhydrous form. In one example, a salt byproduct can
be ammonium sulfate, which can be subsequently used, for example,
as a fertilizer. The isomerization reaction can be carried out at a
pH between about 1.5 and about 6.5 (e.g., 1.5, 1.75, 2, 2.25, 2.5,
2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75,
6, 6.25, 6.5). Preferably, the isomerization reaction can be
carried out at a pH between about 3.5 and about 4.5.
[0029] In certain embodiments, the isomerization reaction is
carried out at a temperature of about 47.degree. C. or greater
(e.g., 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, or higher). Preferably, the isomerization reaction can be
carried out at a temperature of about 60.degree. C. or greater. The
isomerization reaction can be substantially complete within 8,
7.75, 7.5, 7.25, 7, 6.75, 6.5, 6.25, 6, 5.75, 5.5, 5.25, 5, 4.75,
4.5, 4.25, 4, 3.75, 3.5, 3.25, 3, 2.75, 2.5, 2.25, 2, 1.75, 1.5,
1.25, 1, 0.75, 0.5, or 0.25 hours.
[0030] In various embodiments, the isomerization reaction proceeds
substantially without precipitation of cis,trans-muconate from the
reaction mixture. In certain embodiments, the isomerization
reaction includes monitoring the isomerization of cis,cis-muconate
to cis,trans-muconate. In some embodiments, the isomerization
reaction is carried out at a pressure above about atmospheric
pressure.
[0031] In various embodiments, after isomerization, the
cis,trans-muconate can be separated from the solution, medium,
broth, or fermentation broth by further acidification sufficient to
cause the cis,trans-muconic acid to precipitate. The broth can be
acidified to a pH below about 3.0 (e.g., 2.9, 2.8, 2.7, 2.6, 2.5,
2.4, 2.3, 2.2, 2.1, 2, or lower). The broth can be further
acidified to a pH below about 2.
[0032] In certain embodiments, the separating step includes cooling
the solution to a temperature below about 37.degree. C., below
about 25.degree. C., below about -4.degree. C., or below about
-20.degree. C.
[0033] In certain embodiments, the separating step comprises
centrifugation, filtration, or other physical processes for
separating the precipitated cis,trans-muconic acid. In various
embodiments, the separating step includes extracting the
cis,trans-muconate from the fermentation broth using an organic
solvent. The organic solvent can include one or more of methanol,
ethanol, propanol, isopropanol, butanol, acetic acid, acetonitrile,
acetone, and tetrahydrofuran, tert-butyl methyl ether, methyl
tetrahydrofuran, cyclohexanone or cyclohexanol, or mixtures of
these. In one embodiment, the extraction can be carried out at a pH
of between about 7 and 4 (e.g., about 7, 6.75, 6.5, 6.25, 6, 5.75,
5.5, 5.25, 5, 5.75, 5.5, 5.25, 4) without significant precipitation
of the cis,trans-muconic acid, and can include the use of automated
addition of acid to maintain the pH in this region as the
extraction proceeds. In another embodiment, the extraction step can
be carried out at a pH below about 4 (e.g., about 4, 3.75, 3.5,
3.25, 3, 2.75, 2.5, 2.25, 2) in the presence of precipitated
cis,trans-muconic acid which is dissolved by the organic solvent.
In still another embodiment, the extraction step can be performed
without first removing cells, cell debris, proteins, or other
undesired materials from the fermentation broth. In yet another
embodiment, the extraction step can be mediated by a membrane.
[0034] In certain embodiments, the cis,cis-muconate can first be
removed from the fermentation broth, and then subjected to the
isomerization, separation, and purification steps. Such removal can
be accomplished by extraction, precipitation, ion-exchange
chromatography, selective membrane separation, electrodialysis, or
other methods known in the art.
[0035] In some embodiments, the cis,trans-muconic acid is purified
by crystallization using an organic solvent. The organic solvent
can include one or more of methanol, ethanol, propanol,
isopropanol, butanol, acetic acid, acetonitrile, acetone, and
tetrahydrofuran.
[0036] In some embodiments, the crystallization can be performed
without drying the precipitated cis,trans-muconic acid after
recovery from the fermentation broth. In certain embodiments, the
crystallization includes removing an undesired salt from the
separated cis,trans-muconic acid. In various embodiments, the
crystallization includes concentrating the crystallization medium
after collecting a first crop of cis,trans-muconic acid and
collecting a second crop of cis,trans-muconic from the concentrated
medium.
[0037] In certain embodiments, the method for production of
trans,trans-muconate comprises the production of
cis,trans-muconate, isomerizing at least about 65% of the
cis,trans-muconate to trans,trans-muconate, and isolating the
trans,trans-muconate. The method can include isomerizing at least
about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% of the cis,trans-muconate to
trans,trans-muconate. Alternatively, trans,trans-muconate can be
produced or isomerized from cis,cis-muconate under suitable
conditions (e.g., pH, temperature, catalyst, etc.).
[0038] In various embodiments, the isomerized reaction is catalyzed
by I.sub.2, by a precious metal hydrogenation catalyst, by a sponge
metal hydrogenation catalyst, or by a skeletal hydrogenation
catalyst. The precious metal can be any precious metal that
functions as a hydrogenation catalyst (e.g., platinum, palladium,
and the like). The sponge metal or skeletal catalyst can be a
nickel-aluminum alloy (e.g., a RANEY.RTM. nickel catalyst available
from W. R. Grace and Company). The metal catalysts can be in the
form of a heterogeneous catalyst (e.g., particles) or a supported
catalyst (e.g., on a support such as silica, alumina, carbon, and
the like).
[0039] In some embodiments, providing cis,cis-muconate produced
from a renewable carbon source through biocatalytic conversion
employs a bacterial cell transformed with heterologous structural
genes from Klebsiella pneumoniae, which express the enzymes
3-dehydroshikimate dehydratase and protocatechuate decarboxylase,
and from Acinetobacter calcoaceticus, which expresses the enzyme
catechol 1,2-dioxygenase, wherein a culture of the bacterial cell
biocatalytically converts glucose to cis,cis-muconic acid at a rate
at least sufficient to convert 1.38M glucose to at least about 0.42
M cis,cis-muconic acid within about 88 hours. The bacterial cell
transformant can include heterologous DNA sequences which express
the enzymes 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase
and 3-dehydroquinate synthase. The bacterial cell transformant can
includes heterologous DNA sequences which express the enzymes
transketolase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase
and 3-dehydroquinate synthase. The bacterial cell can be selected
from mutant cell lines having mutations in the common pathway of
aromatic amino acid biosynthesis that block conversion of
3-dehydroshikimate to chorismate. The bacterial cell is selected
from mutant cell lines having mutations in the common pathway of
aromatic amino acid biosynthesis that block conversion of
3-dehydroshikimate to chorismate.
[0040] In certain embodiments, providing cis,cis-muconate produced
from a renewable carbon source through biocatalytic conversion
includes culturing a bacterial cell transformed with structural
genes from Klebsiella pneumoniae which express the enzyme species
3-dehydroshikimate dehydratase and protocatechuate decarboxylase,
and a structural gene from Acinetobacter calcoaceticus which
expresses the enzyme species catechol 1,2-dioxygenase, in a medium
containing a carbon source which is converted to 3-dehydroshikimate
by the enzymes in the common pathway of aromatic amino acid
biosynthesis of the cell, to produce cis,cis-muconic acid at a rate
of at least about 0.95 millimoles/liter/hour, by the biocatalytic
conversion of 3-dehydroshikimate. In other embodiments,
cis,cis-muconic acid is produced at a rate of at least about 0.97,
1.0, 1.2, 1.4, 1.6, 1.8, 2.0 millimoles/liter/hour or greater.
[0041] In various embodiments, providing cis,cis-muconate produced
from a renewable carbon source through biocatalytic conversion
comprises culturing a transformed bacterial cell, which expresses
heterologous structural genes encoding 3-dehydroshikimate
dehydratase, protocatechuate decarboxylase, catechol
1,2-dioxygenase, transketolase, 3-deoxy-D-arabino-heptulosonate
7-phosphate synthase, and 3-dehydroquinate synthase, in a medium
containing a carbon source which is converted to
3-dehydroshikimate, by the enzymes in the common pathway of
aromatic amino acid biosynthesis of the cell, to produce
cis,cis-muconic acid at a rate of at least about 0.95
millimoles/liter/hour by the biocatalytic conversion of
3-dehydroshikimate. In other embodiments, cis,cis-muconic acid is
produced at a rate of at least about 0.97, 1.0, 1.2, 1.4, 1.6, 1.8,
2.0 millimoles/liter/hour or greater.
[0042] In some embodiments, providing cis,cis-muconate produced
from a renewable carbon source through biocatalytic conversion
comprises culturing a bacterial cell, transformed with structural
genes from Klebsiella pneumoniae which express the enzyme species
3-dehydroshikimate dehydratase and protocatechuate decarboxylase
and a structural gene from Acinetobacter calcoaceticus which
expresses the enzyme catechol 1,2-dioxygenase in a medium
containing a carbon source, under conditions in which the carbon
source is biocatalytically converted to cis,cis-muconic acid at a
rate of at least about 0.95 millimoles/liter/hour. In other
embodiments, cis,cis-muconic acid is produced at a rate of at least
about 0.97, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 millimoles/liter/hour or
greater.
[0043] Other aspects and advantages of the invention will become
apparent from the following drawings and description, all of which
illustrate principles of the invention, by way of example only.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The advantages of the invention described above, together
with further advantages, can be better understood by referring to
the following description taken in conjunction with the
accompanying drawings. The drawings are not necessarily to scale,
emphasis instead generally being placed upon illustrating the
principles of the invention.
[0045] FIG. 1 shows the common pathway of aromatic amino acid
biosynthesis and the divergent pathway synthesizing cis,cis-muconic
acid from 3-dehydroshikimate.
[0046] FIG. 2 shows a plasmid map of plasmid p2-47 and illustrates
how plasmid pKD8.243A can be generated from plasmids p2-47,
pSU1-31, and pSUaroZY 157-27.
[0047] FIG. 3 shows a plasmid map of pKD8.292 and illustrates how
plasmid pKD8.292 can be generated from plasmids pIB1345 and
pCL1920.
[0048] FIGS. 4A and 4B show examples of .sup.1H NMR spectra of
cis,trans-muconic acid in different media.
[0049] FIG. 5 shows examples of .sup.1H NMR traces for a muconate
isomerization reaction at pH 7.
[0050] FIG. 6 shows examples of .sup.1H NMR traces for a muconate
isomerization reaction at pH 4.
[0051] FIG. 7 shows examples of HPLC traces for a muconate
isomerization reaction at pH 7.
[0052] FIG. 8 shows examples of HPLC traces for a muconate
isomerization reaction at pH 4.
[0053] FIG. 9 shows examples of a time course for a muconate
isomerization reaction at pH 7.
[0054] FIG. 10 shows examples of a time course for a muconate
isomerization reaction at pH 4.
[0055] FIG. 11 shows a batch cultivation production of muconic
acid.
DETAILED DESCRIPTION OF THE INVENTION
[0056] The invention includes methods for producing
cis,trans-muconic acid and trans,trans-muconic acid from
fermentable carbon sources capable of being used by a host cell
having a common pathway of aromatic amino acid biosynthesis, for
example, one which is functional through to the intermediate DHS,
plus the ability to express the enzymes aroZ, aroY, and catA. In
one preferred embodiment, the method comprises the steps of
culturing the host cell in the presence of a fermentable carbon
source to produce cis,cis-muconic acid, and isomerizing the
cis,cis-muconic acid to produce cis,trans-muconic acid or
trans,trans-muconic acid.
[0057] Fermentable carbon sources can include essentially any
carbon source capable of being biocatalytically converted into
D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP), two
precursor compounds to the common pathway of aromatic amino acid
biosynthesis. Suitable carbon sources include, but are not limited
to, biomass-derived, or renewable, sources such as starches,
cellulose, and sugar moieties such as glucose, pentoses, and
fructose, as well as other carbon sources capable of supporting
microbial metabolism, for example, carbon monoxide. In one
embodiment, D-glucose can be used as the biomass-derived carbon
source.
[0058] Host cells suitable for use in the present invention include
members of genera that can be utilized for biosynthetic production
of desired aromatic compounds. In some embodiments, such host cells
are suitable for industrial-scale biosynthetic production of
desired aromatic compounds. In particular, suitable host cells can
have an endogenous common pathway of aromatic amino acid
biosynthesis that is functional at least to the production of DHS.
Common aromatic pathways are endogenous in a wide variety of
microorganisms, and can be used for the production of various
aromatic compounds. For example, microbial aromatic amino acid
biosynthesis pathways as described in U.S. Pat. Nos. 5,168,056 and
5,616,496, the disclosures of both of which are incorporated herein
by reference in their entirety, can be utilized in the present
invention.
[0059] FIG. 1 shows the common pathway of aromatic amino acid
biosynthesis and the divergent pathway synthesizing cis,cis-muconic
acid from 3-dehydroshikimate through the common aromatic pathway
that leads from E4P and PEP to chorismic acid with many
intermediates in the pathway. The availability of E4P can be
increased by the pentose phosphate pathway enzyme transketolase,
encoded by the tkt gene. The intermediates in the pathway include
3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP),
3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS), shikimic acid,
shikimate 3-phosphate (S3P), and
5-enolpyruvoylshikimate-3-phosphate (EPSP). The enzymes in the
common pathway include DAHP synthase (aroF), DHQ synthase (aroB),
DHQ dehydratase (aroD), shikimate dehydrogenase (aroE), shikimate
kinase (aroL, aroK), EPSP synthase (aroA) and chorismate synthase
(aroC).
[0060] Host cells including common pathways of this type include
prokaryotes belonging to the genera Escherichia, Klebsiella,
Corynebacterium, Brevibacterium, Arthrobacter, Bacillus,
Pseudomonas, Streptomyces, Staphylococcus, and Serratia. Eukaryotic
host cells can also be utilized, for example, with yeasts of the
genus Saccharomyces or Schizosaccharomyces.
[0061] More specifically, prokaryotic host cells can be derived
from species that include Escherichia coli, Klebsiella pneumonia,
Corynebacterium glutamicum, Corynebacterium herculis,
Brevibacterium divaricatum, Brevibacterium lactofermentum,
Brevibacterium flavum, Bacillus brevis, Bacillus cereus, Bacillus
circulans, Bacillus coagulans, Bacillus lichenformis, Bacillus
megaterium, Bacillus mesentericus, Bacillus pumilis, Bacillus
subtilis, Pseudomonas aeruginosa, Pseudomonas angulata, Pseudomonas
fluorescens, Pseudomonas tabaci, Streptomyces aureofaciens,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, Streptomyces kasugensis, Streptomyces lavendulae,
Streptomyces lipmanii, Streptomyces lividans, Staphylococcus
epidermis, Staphylococcus saprophyticus, and Serratia marcescens.
Examples of eukaryotic host cells include Saccharomyces cerevisiae
and Saccharomyces carlsbergensis.
[0062] Host cells can include auxotrophic mutant cell lines having
a mutation that blocks the conversion of DHS to the branch point
molecule, chorismate. Such mutants are unable to catalyze the
conversion of 3-dehydroshikimate (DHS) into chorismate due to a
mutation in one or more of the genes encoding shikimate
dehydrogenase, shikimate kinase, EPSP synthase and chorismate
synthase, and will thus accumulate elevated intracellular levels of
DHS. Examples of such mutant cell lines include Escherichia coli
strains AB2834, AB2829 and AB2849.
[0063] E. coli AB2834 is unable to catalyze the conversion of
3-dehydroshikimate (DHS) into shikimic acid due to a mutation in
the aroE locus which encodes shikimate dehydrogenase. Use of E.
coli AB2834 can ensure that the carbon flow directed into aromatic
amino acid biosynthesis is not processed beyond DHS. Similarly E.
coli AB2829 (which is unable to catalyze the conversion of
shikimate 3-phosphate (S3P) into 5-enoipyruvylshikimate-3-phosphate
(EPSP) due to a mutation in the aroA locus which encodes EPSP
synthase) and E. coli AB2849 (which is unable to catalyze the
conversion of EPSP into chorismic acid due to a mutation in the
aroC locus which encodes chorismate synthase) also result in
increased intracellular levels of DHS.
[0064] Host cells can be transformed so that the intracellular DHS
can be used as a substrate for biocatalytic conversion to catechol,
which can thereafter be converted to muconic acid. For example,
host cells can be transformed with recombinant DNA to force carbon
flow away from the common pathway of aromatic amino acid
biosynthesis after DHS is produced and into a divergent pathway to
produce muconic acid.
[0065] As shown in FIG. 1, the intermediates in the divergent
pathway are protocatechuate, catechol, and cis,cis-muconic acid.
The enzyme responsible for the biocatalytic conversion of DHS to
protocatechuate is the enzyme 3-dehydroshikimate dehydratase,
labeled "aroZ" in FIG. 1. The enzyme responsible for the
decarboxylation of protocatechuate to form catechol is
protocatechuate decarboxylase, labeled "aroY" in FIG. 1. Lastly,
the enzyme catalyzing the oxidation of catechol to produce
cis,cis-muconic acid is catechol 1,2-dioxygenase, labeled "catA" in
FIG. 1. In accordance with standard notation, the genes for the
expression of these enzymes are denoted using italics and are thus
aroZ, aroY, and catA respectively. The cis,cis-muconic acid can
subsequently be isomerized (not shown). In one embodiment of the
invention, host cells may exhibit constitutive expression of the
genes aroZ, aroY, and catA. In another embodiment, host cells may
exhibit constitutive expression of any one or more of the genes
aroZ, aroY and catA; or any combination of two thereof. In yet
another embodiment, host cells may exhibit constitutive expression
of none of aroZ, aroY and catA.
[0066] The enzymes 3-dehydroshikimate dehydratase and
protocatechuate decarboxylase are recruited from the ortho cleavage
pathways which enable microbes such as Neurospora, Aspergillus,
Acinetobacter, Klebsiella, and Pseudomonas to use aromatics
(benzoate and p-hydroxybenzoate) as well as hydroaromatics
(shikimate and quinate) as sole sources of carbon for growth. DHS
dehydratase plays a critical role in microbial catabolism of quinic
and shikimic acid. Protocatechuate decarboxylase was formulated by
Patel to catalyze the conversion of protocatechuate into catechol
during catabolism of p-hydroxybenzoate by Klebsiella aerogenes.
Reexamination of Patel's strain (now referred to as Enterobacter
aerogenes) [(a) Grant, D. J. W.; Patel, J. C. Antonie van
Leewenhoek 1969, 35, 325. (b) Grant, D. J. W. Antonie van
Leewenhoek 1970, 36, 161] recently led Ornston to conclude that
protocatechuate decarboxylase was not metabolically significant in
catabolism of p-hydroxybenzoate [Doten, R. C.; Ornston, N. J.
Bacteriol. 1987, 169, 5827].
[0067] A mechanism for transforming the host cell to direct carbon
flow into the divergent pathway can involve the insertion of
genetic elements including expressible sequences coding for
3-dehydroshikimate dehydratase, protocatechuate decarboxylase, and
catechol 1,2-dioxygenase. Regardless of the exact mechanism
utilized, it is contemplated that the expression of these enzymatic
activities will be effected or mediated by the transfer of
recombinant genetic elements into the host cell. Genetic elements
as herein defined include nucleic acids (generally DNA and RNA)
having expressible coding sequences for products such as proteins,
apoproteins, or antisense RNA, which can perform or control pathway
enzymatic functions. The expressed products can function as
enzymes, repress or derepress enzyme activity, or control
expression of enzymes. The nucleic acids coding these expressible
sequences can be either chromosomal (e.g., inserted or integrated
into a host cell chromosome) or extrachromosomal (e.g., carried by
plasmids, cosmids, etc.).
[0068] The genetic elements of the present invention can be
introduced into a host cell by plasmids, cosmids, phages, yeast
artificial chromosomes or other vectors that mediate transfer of
the genetic elements into a host cell. These vectors can include an
origin of replication along with cis-acting control elements that
control replication of the vector and the genetic elements carried
by the vector. Selectable markers can be present on the vector to
aid in the identification of host cells into which the genetic
elements have been introduced. For example, selectable markers can
be genes that confer resistance to particular antibiotics such as
tetracycline, ampicillin, chloramphenicol, kanamycin, or
neomycin.
[0069] Introducing genetic elements into a host cell can utilize an
extrachromosomal multi-copy plasmid vector into which genetic
elements are inserted. Plasmid borne introduction of the genetic
element into host cells involves an initial cleaving of a plasmid
with a restriction enzyme, followed by ligation of the plasmid and
genetic elements in accordance with the invention. Upon
recircularization of the ligated recombinant plasmid, transduction
or other mechanism (e.g., electroporation, microinjection, and the
like) for plasmid transfer is utilized to transfer the plasmid into
the host cell. Plasmids suitable for insertion of genetic elements
into the host cell include, but are not limited to, pBR322, and its
derivatives such as pAT153, pXf3, pBR325, pBr327, pUC vectors,
pACYC and its derivatives, pSC101 and its derivatives, and ColE1.
In addition, cosmid vectors such as pLAFR3 are also suitable for
the insertion of genetic elements into host cells. Examples of
plasmid constructs include, but are not limited to, p2-47,
pKD8.243A, pKD8.243B, and pSUaroZY157-27, which carry the aroZ and
aroY loci isolated from Klebsiella pneumoniae, which respectively
encode 3-dehydroshikimate dehydratase and protocatechuate
decarboxylase. Additional examples of plasmid constructs include
pKD8.292, which carries genetic fragments endogenous to
Acinetobacter calcoaceticus catA, encoding catechol
1,2-dioxygenase.
[0070] Methods for transforming a host cell can also include
insertion of genes encoding for enzymes, which increase commitment
of carbon into the common pathway of aromatic amino acid
biosynthesis. The expression of a gene is primarily directed by its
own promoter, although other genetic elements including optional
expression control sequences such as repressors, and enhancers can
be included to control expression or derepression of coding
sequences for proteins, apoproteins, or antisense RNA. In addition,
recombinant DNA constructs can be generated whereby the gene's
natural promoter is replaced with an alternative promoter to
increase expression of the gene product. Promoters can be either
constitutive or inducible. A constitutive promoter controls
transcription of a gene at a constant rate during the life of a
cell, whereas an inducible promoter's activity fluctuates as
determined by the presence (or absence) of a specific inducer. For
example, control sequences can be inserted into wild type host
cells to promote overexpression of selected enzymes already encoded
in the host cell genome, or alternatively can be used to control
synthesis of extrachromosomally encoded enzymes.
[0071] Control sequences to promote overproduction of DHS can be
used. As previously noted, DHS is synthesized in the common pathway
by the sequential catalytic activities of the tyrosine-sensitive
isozyme of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP)
synthase (encoded by aroF) and 3-dehydroquinate (DHQ) synthase
(encoded by aroB) along with the pentose phosphate pathway enzyme
transketolase (encoded by tkt). The expression of these
biosynthetic enzymes can be amplified to increase the conversion of
D-glucose into DHS. Increasing the in vivo catalytic activity of
DAHP synthase, the first enzyme of the common pathway, increases
the flow of D-glucose equivalents directed into aromatic
biosynthesis. However, levels of DAHP synthase catalytic activity
are reached beyond which no further improvements are achieved in
the percentage of D-glucose that is committed to aromatic
biosynthesis. At this limiting level of aromatic amino acid
biosynthesis, amplification of the catalytic levels of the pentose
phosphate pathway enzyme transketolase achieves sizable increases
in the percentage of D-glucose siphoned into the pathway.
[0072] Amplified transketolase activity can increase D-erythrose
4-phosphate concentrations. As one of the two substrates for DAHP
synthase, limited D-erythrose 4-phosphate availability can limit
DAHP synthase catalytic activity. Therefore, one method for
amplifying the catalytic activities of DAHP synthase, DHQ synthase
and DHQ dehydratase is to overexpress the enzyme species by
transforming the microbial catalyst with a recombinant DNA sequence
encoding these enzymes.
[0073] Amplified expression of DAHP synthase and transketolase can
create a surge of carbon flow directed into the common pathway of
aromatic amino acid biosynthesis, which is in excess of the normal
carbon flow directed into this pathway. If the individual rates of
conversion of substrate into product catalyzed by individual
enzymes in the common aromatic amino acid pathway are less than the
rate of DAHP synthesis, the substrates of these rate-limiting
enzymes can accumulate intracellularly.
[0074] Microbial organisms such as E. coli frequently cope with
accumulated substrates by exporting such substrates into the
external environment, such as the bulk fermentation medium. This
results in a loss of carbon flow through the common pathway since
exported substrates are typically lost to the microbe's metabolism.
DHQ synthase is an example of a rate-limiting common pathway
enzyme. Amplified expression of DHQ synthase removes the
rate-limiting character of this enzyme, and prevents the
accumulation of DAHP and its nonphosphorylated analog, DAH. DHQ
dehydratase is not rate-limiting. Therefore, amplified expression
of aroF-encoded DAHP synthase, tkt-encoded transketolase and
aroB-encoded DHQ synthase increases production of DHS, which in the
presence of DHS dehydratase and protocatechuate decarboxylase is
converted to catechol, which is subsequently biocatalytically
converted to cis,cis-muconic acid, which can subsequently be
isomerized.
[0075] One plasmid that can promote the efficiency of carbon flow
along the common pathway between the carbon source and DHS is
plasmid pKD136, which encodes the aroF, tkt and aroB genes. Plasmid
pKD136-directs the surge of carbon flow into aromatic biosynthesis
due to amplified expression of DAHP synthase (encoded by aroF) and
transketolase (encoded by tkt). This surge of carbon flow is then
delivered intact into DHS synthesis by pKD136 due to amplified
expression of DHQ synthase (encoded by aroB).
[0076] Thus, as a preferred embodiment of the present invention, a
heterologous strain of Escherichia coli expressing genes encoding
DHS dehydratase, protocatechuate decarboxylase, and catechol
1,2-dioxygenase was constructed enabling the biocatalytic
conversion of D-glucose to cis,cis-muconic acid. Efficient
conversion of D-glucose to DHS was accomplished upon transformation
of the host cell with pKD136. The strain E. coli AB2834/pKD136 was
then transformed with plasmids pKD8.243A and pKD8.292. The result
was E. coli AB2834/pKD136/pKD8.243A/pKD8.292 that expresses the
enzymes 3-dehydroshikimate dehydratase (aroZ), protocatechuate
decarboxylase (aroY) and catechol 1,2-dioxygenase (catA). This
bacterial cell line was deposited with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville Md. 20852, on Aug. 1,
1995 and assigned accession number 69875.
[0077] In another embodiment, E. coli AB2834/pKD136 is transformed
with plasmids p2-47 and pKD8.292 to generate E. coli
AB2834/pKD136/p2-47/pKD8.292. In another embodiment, E. coli
AB2834/pKD136 is transformed with plasmids pKD8.243B and pKD8.292
to generate E. coli AB2834/pKD136/p2-47/pKD8.292. Each of these
heterologous host cell lines catalyzes the conversion of D-glucose
into cis,cis-muconic acid. Synthesized cis,cis-muconic acid
accumulates extracellularly and can be separated from the cells.
Subsequently, the cis,cis-muconic acid can be isomerized into
cis,trans-muconic acid and further to trans,trans-muconic acid as
desired.
[0078] The present invention thus relates to a transformant of a
host cell having an endogenous common pathway of aromatic amino
acid biosynthesis. The transformant is characterized by the
constitutive expression of heterologous genes encoding
3-dehydroshikimate dehydratase, protocatechuate decarboxylase, and
catechol 1,2-dioxygenase. In one embodiment, the cell transformant
is further transformed with expressible recombinant DNA sequences
encoding the enzymes transketolase, DAHP synthase, and DHQ
synthase. In another embodiment, the host cell is selected from the
group of mutant cell lines including mutations having a mutation in
the common pathway of amino acid biosynthesis that blocks the
conversion of 3-dehydroshikimate to chorismate. In yet another
embodiment, the genes encoding 3-dehydroshikimate dehydratase and
protocatechuate decarboxylase are endogenous to Klebsiella
pneumoniae. In a further embodiment, the heterologous genes
encoding catechol 1,2-dioxygenase are endogenous to Acinetobacter
calcoaceticus.
Renewable Muconate
[0079] Muconic acids produced from renewable, biologically derived
carbon sources will be composed of carbon from atmospheric carbon
dioxide which has been incorporated by plants (e.g., from a carbon
source such as glucose, sucrose, glycerin, or plant oils).
Therefore, such muconic acids include renewable carbon rather than
fossil fuel-based or petroleum-based carbon in their molecular
structure. Accordingly, the biosynthetic muconate that is the
subject of this patent, and associated derivative products, will
have a smaller carbon footprint than muconate and associated
products produced by conventional methods because they do not
deplete fossil fuel or petroleum reserves and because they do not
increase the amount of carbon in the carbon cycle (e.g., life cycle
analysis shows no net carbon increase to the global carbon
balance).
[0080] The biosynthetic muconate and associated products can be
distinguished from muconate and associated products produced from a
fossil fuel or petrochemical carbon source by methods known in the
art, such as dual carbon-isotopic finger printing. This method can
distinguish otherwise chemically-identical materials, and
distinguishes carbon atoms in the material by source, that is
biological versus non-biological, using the .sup.14C and .sup.13C
isotope ratios. The carbon isotope .sup.14C is unstable, and has a
half life of 5730 years. Measuring the relative abundance of the
unstable .sup.14C isotope relative to the stable .sup.13C isotope
allows one to distinguish specimen carbon between fossil (long
dead) and biospheric (alive and thus renewable) feedstocks (See
Currie, L. A. "Source Apportionment of Atmospheric Particles,"
Characterization of Environmental Particles, J. Buffle and H. P.
van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental
Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3-74).
The basic assumption in radiocarbon dating is that the constancy of
.sup.14C concentration in the atmosphere leads to the constancy of
.sup.14C in living organisms.
[0081] When dealing with an isolated sample, the age of a sample
can be deduced approximately by the relationship
t=(-5730/0.693)ln(A/A.sub.o) where t=age, 5730 years is the
half-life of the unstable .sup.14C isotope, and A and A.sub.o are
the specific .sup.14C activity of the sample and of the modern
standard, respectively (Hsieh, Y., Soil ScL Soc. Am J., 56, 460,
(1992)). However, because of atmospheric nuclear testing since 1950
and the burning of fossil fuel since 1850, .sup.14C has acquired a
second, geochemical time characteristic. Its concentration in
atmospheric CO.sub.2, and hence in the living biosphere,
approximately doubled at the peak of nuclear testing, in the
mid-1960s. It has since been gradually returning to the
steady-state cosmogenic (atmospheric) baseline isotope rate
(.sup.14C/.sup.12C) of ca. 1.2.times.10.sup.-12, with an
approximate relaxation half-life of 7-10 years. (This latter
half-life must be distinguished from the isotopic half-life, that
is, one must use the detailed atmospheric nuclear input/decay
function to trace the variation of atmospheric and biospheric
.sup.14C since the onset of the nuclear age.) It is this latter
biospheric .sup.14C time characteristic that holds out the promise
of annual dating of recent biospheric carbon. .sup.14C can be
measured by accelerator mass spectrometry (AMS), with results given
in units of fraction of modern carbon (f.sub.M). f.sub.M is defined
by National Institute of Standards and Technology (NIST) Standard
Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids
standards HOxI and HOxII, respectively. The fundamental definition
relates to 0.95 times the .sup.14C/.sup.12C isotope ratio HOxI
(referenced to AD 1950). For the current living biosphere (plant
material), f.sub.M.apprxeq.1.1.
[0082] The ratio of the stable carbon isotopes .sup.13C and
.sup.12C provides a complementary route to source discrimination
and apportionment. The .sup.13C/.sup.12C ratio in a given
biosourced material is a consequence of the .sup.13C/.sup.12C ratio
in atmospheric carbon dioxide at the time the carbon dioxide is
fixed and also reflects the precise metabolic pathway. Regional
variations also occur. Petroleum, C.sub.3 plants (the broadleaf),
C.sub.4 plants (the grasses), and marine carbonates all show
significant differences in .sup.13C/.sup.12C and the corresponding
.delta. .sup.13C values. Furthermore, lipid matter of C.sub.3 and
C.sub.4 plants analyze differently than materials derived from the
carbohydrate components of the same plants as a consequence of the
metabolic pathway. Within the precision of measurement, .sup.13C
shows large variations due to isotopic fractionation effects, the
most significant of which for the instant invention is the
photosynthetic mechanism. The major cause of differences in the
carbon isotope ratio in plants is closely associated with
differences in the pathway of photosynthetic carbon metabolism in
the plants, particularly the reaction occurring during the primary
carboxylation (e.g., the initial fixation of atmospheric CO.sub.2).
Two large classes of vegetation are those that incorporate the
C.sub.3 (or Calvin-Benson) photosynthetic cycle and those that
incorporate the C.sub.4 (or Hatch-Slack) photosynthetic cycle.
C.sub.3 plants, such as hardwoods and conifers, are dominant in the
temperate climate zones. In C.sub.3 plants, the primary CO.sub.2
fixation or carboxylation reaction involves the enzyme
ribulose-1,5-diphosphate carboxylase and the first stable product
is a 3-carbon compound. C.sub.4 plants, on the other hand, include
such plants as tropical grasses, corn and sugar cane. In C.sub.4
plants, an additional carboxylation reaction involving another
enzyme, phosphoenol-pyruvate carboxylase, is the primary
carboxylation reaction. The first stable carbon compound is a
4-carbon acid, which is subsequently decarboxylated. The CO.sub.2
thus released is refixed by the C.sub.3 cycle.
[0083] Both C.sub.4 and C.sub.3 plants exhibit a range of
.sup.13C/.sup.12C isotopic ratios, but typical values are ca. -10
to -14 per mil (C.sub.4) and -21 to -26 per mil (C.sub.3) (Weber et
al., J. Agric. Food Chem., 45, 2942 (1997)). Coal and petroleum
fall generally in this latter range. The .sup.13C measurement scale
was originally defined by a zero set by pee dee belemnite (PDB)
limestone, where values are given in parts per thousand deviations
from this material. The .delta..sup.13C values are in parts per
thousand (per mil), abbreviated %.sub.o, and are calculated as
follows:
.delta..sup.13C.ident.(.sup.13C/.sup.12C)sample-(.sup.13C/.sup.12C)stand-
ard/(.sup.13C/.sup.12C)standard.times.1000%.sub.o
Since the PDB reference material (RM) has been exhausted, a series
of alternative RMs have been developed in cooperation with the
IAEA, USGS, NIST, and other selected international isotope
laboratories. Notations for the per mil deviations from PDB is
.delta..sup.13C. Measurements are made on CO.sub.2 by high
precision stable ratio mass spectrometry (IRMS) on molecular ions
of masses 44, 45 and 46.
[0084] Therefore, the biosynthesized muconate and compositions
including biosynthesized muconate can be distinguished from their
fossil-fuel and petrochemical derived counterparts on the basis of
.sup.14C (f.sub.M) and dual carbon-isotopic fingerprinting,
indicating new compositions of matter (e.g., U.S. Pat. Nos.
7,169,588, 7,531,593, and 6,428,767). The ability to distinguish
these products is beneficial in tracking these materials in
commerce. For example, products comprising both new and old carbon
isotope profiles can be distinguished from products made only of
old materials. Hence, the biosynthetic muconate and derivative
materials can be followed in commerce on the basis of their unique
profile.
EXAMPLES
Example 1
Cloning of the aroZ Gene
[0085] The gene encoding DHS dehydratase, designated aroZ, was
isolated from a genomic library of Klebsiella pneumoniae DNA.
Genomic DNA was purified from K. pneumoniae strain A170-40 and
partially digested with BamH I to produce fragments in the range of
15 kb to 30 kb. The resulting DNA fragments were ligated to cosmid
pLAFR3 which had previously been digested with BamH I and
subsequently treated with calf intestinal alkaline phosphatase.
pLAFR3 is a tetracycline resistant cosmid possessing the RK2
replicon. Ligated DNA was packaged using Packagene Packaging System
(Promega), and the resulting phage particles were used to infect E.
coli DH5.alpha./pKD136. Plasmid pKD136 is a pBR325-based vector
(pMB1 origin of replication) containing genes which encode
transketolase (tkt), DAHP synthase (aroF), and DHQ synthase (aroB)
as well as an ampicillin resistance gene. Colonies which were
resistant to both tetracycline and ampicillin were subsequently
plated onto chromogenic minimal medium (M9) plates containing
D-glucose (4 g L), shikimic acid (0.04 g L), ferric citrate (0.07 g
L), p-toluidine (1.9 g L), ampicillin (0.05 g L), and tetracycline
(0.013 g L). After incubation at 37.degree. C. for 48 h, the growth
medium surrounding colony 5-87 appeared brown in color, analogous
to the darkening of the medium that occurred when protocatechuic
acid was spotted onto the plate. DNA was purified from a culture of
colony 5-87 and consisted of pKD136 and a tetracycline resistant
cosmid referred to as p5-87. Cosmid p5-87 contained a 14 kb BamH I
fragment which when digested to completion with BamH I produced
four detectable fragments of DNA.
Example 2
Confirmation of the Cloning of the aroZ Gene
[0086] Confirmation that cosmid p5-87 contained the aroZ gene
relied on the fact that transformation of an E. coli strain which
typically converts D-glucose into DHS could further convert DHS
into protocatechuic acid. E. coli AB2834 accumulates DHS in the
culture supernatant due to a mutation in the aroE gene, which
encodes shikimate dehydrogenase. Conversion of D-glucose to DHS is
maximized when AB2834 is transformed with pKD136. AB2834 was
co-transformed with pKD136 and p5-87 to produce colonies that were
resistant to both ampicillin and tetracycline. One liter of LB
medium (4 L Erlenmeyer flask) was inoculated with an overnight
culture (5 mL) of AB2834/pKD136/p5-87. The culture was grown at
37.degree. C. for 8 h with agitation (250 rpm). The cells were then
harvested and resuspended in one liter (4 L Erlenmeyer flask) of
minimal M9 medium containing glucose (10 g L), shikimic acid (0.04
g L), ampicillin (0.05 g L), and tetracycline (0.013 g L). The
culture was returned to 37.degree. C. incubation. Aliquots of the
culture were removed after 24 h and 64 h and centrifuged to remove
cells. Five milliliters of isolated supernatant was collected from
each sample and the water was removed in vacuo. Samples were
redissolved in D.sub.2O and concentrated in vacuo. Repetition of
this procedure resulted in exchange of residual water with D.sub.2O
and samples suitable for analysis by .sup.1H NMR. Using the sodium
salt of 3-(trimethylsilyl)ProPionic-2,2,3,3-d.sub.4 acid as an
internal standard, it was determined that approximately 9 mM
protocatechuic acid had accumulated in the culture supernatant.
Diagnostic resonances at .delta.6.94 (d, 7 Hz, 1H) and .delta. 7.48
(d, 7 Hz, 2H) were indicative of protocatechuic acid. DHS was not
detected in the culture supernatant. It was concluded from this
experiment that the gene encoding DHS dehydratase (aroZ) was
localized on plasmid p5-87.
Example 3
Subcloning of the aroZ Gene
[0087] In an effort to minimize the size of the aroZ-encoding
insert plasmid p5-87 was digested with BamH I and the resulting
fragments were ligated to vector pSU19 which had previously been
digested with BamH I and treated with phosphatase. Plasmid pSU19
contains the p15A replicon and the gene which imparts resistance to
chloramphenicol. Following transformation of the ligation products
into E. coli DH5.alpha./pKD136, the resulting ampicillin and
chloramphenicol resistant colonies were screened as described in
Example 1 for the ability to turn chromogenic minimal medium
agarose plates containing p-toluidine and ferric citrate brown.
Using this technique, plasmid pSU1-31 was isolated which consisted
of a 3.5 kb BamH I insert contained in pSU19. When
AB2834/pKD136/pSU1-31 was grown on a 1 L scale under conditions
similar to those described in Example 1, .sup.1H NMR analysis of
the culture supernatant of indicated that 11 mM protocatechuic acid
accumulated extracellularly.
Example 4
Cloning of the aroY Gene
[0088] A fragment of DNA containing the aroY gene was isolated
based on the fact that a strain which normally synthesizes
protocatechuate will instead synthesize catechol in the presence of
catalytically active protocatechuate decarboxylase. Cosmid p4-20
was prepared which contained the 3.5 kb BamH I aroZ fragment
localized in pLAFR3. A library of Klebsiella pneumoniae DNA
digested with EcoR I was prepared in cosmid p4-20 analogous to what
had been constructed earlier in pLAFR3. DNA packaged in lambda
phage heads was used to infect E. coli DH5.alpha./pKD136, resulting
in colonies resistant to both ampicillin and tetracycline. Colonies
were screened on chromogenic minimal medium agarose plates
containing p-toluidine and ferric citrate. Since addition of
catechol to chromogenic minimal medium gives rise to a more intense
darkening of the surrounding agarose than the addition of an equal
quantity of protocatechuic acid, it was expected that those
colonies synthesizing catechol could be selected from a background
of colonies synthesizing protocatechuate. After incubation at
37.degree. C. for approximately 24 h, colony 2-47 was producing a
local region of brown that was lacking from all other colonies.
[0089] Isolation of DNA from colony 2-47 yielded plasmid pKD136 and
plasmid p2-47 which were subsequently co-transformed into competent
cells to yield E. coli AB2834/pKD136/p2-47. The culture supernatant
of AB2834/pKD136/p2-47 was analyzed by .sup.1H NMR as described in
Example 2. After 48 h in minimal medium, a solution of 56 mM
D-glucose was converted to a solution of 20 mM catechol by
AB2834/pKD136/p2-47.
Example 5
Subcloning of the aroY Gene
[0090] Similar to the original strategy for isolation of the DNA
encoding protocatechuate decarboxylase, subcloning of the aroY EcoR
I fragment to its minimal size also relied on synthesis of catechol
by an aroE host strain in the presence of DHS dehydratase.
Digestion of p2-47 to completion with EcoR I indicated that the
aroY insert consisted of two EcoR I fragments of approximately 8 kb
and 11.9 kb. Localization of the 11.9 kb EcoR I fragment in pSU1-31
yielded plasmid pSUaroZY157-27. When grown on a 1 L scale under
conditions similar to those described in Example 2, E. coli
AB2834/pKD136/pSUaroZY157-27 accumulated 16 mM catechol in the
culture supernatant when supplied with 56 mM D-glucose. Mapping of
the 11.9 kb EcoR I fragment in conjunction with further subcloning
indicated that the aroY gene was likely located near the middle of
the 11.9 kb fragment. Digestion of pSUaroZY157-27 with Hind III
produced a 2.3 kb Hind III fragment which was inserted into
pSU1-31, yielding plasmid pKD8.243A (FIG. 2). Plasmid pKD8.243B in
which the 2.3 kb Hind III fragment is in the opposite orientation
relative to the vector was also isolated. Each of these plasmids
was co-transformed into AB2834 with plasmid pKD136. When grown on a
1 L scale under conditions similar to those described in Example 2,
AB2834/pKD136/pKD8.243A synthesized 16 mM catechol from 56 mM
D-glucose within 48 h whereas AB2834/pKD136/pKD8.243B synthesized
10 mM catechol. Protocatechuic acid (<4 mM) was also detected in
some of the culture supernatants, though not on a consistent basis
and not always at the end of the microbial synthesis. Bacterial
cell line AB2834/pKD136/pKD8.243A, which expresses the enzyme
species 3-dehydroshikimate dehydratase and protocatechuate
decarboxylase, was deposited with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville Md. 20852, on Mar. 19,
1996 was assigned accession number 98014.
Example 6
Enzymatic Activities of DHS Dehydratase, Protocatechuate
Decarboxylase, and Catechol 1,2-Dioxygenase
[0091] Expression of catechol 1,2-dioxygenase in an organism
capable of catalyzing conversion of D-glucose into catechol was
expected to result in microbial synthesis of cis,cis-muconic acid.
Plasmid pIB 1345 was obtained which contains the Acinetobacter
calcoaceticus catA gene expressed from a lac promoter supplied by
the host vector pUC19. A three plasmid system was designed for
microbial synthesis of cis,cis-muconate from D-glucose. Plasmids
pKD136 (pMB1 origin, ampicillin resistance) and pKD8.243A (p15A
origin, chloramphenicol resistance) were found to be stably
maintained under the growth conditions employed. A third plasmid,
pCL1920, was chosen for expression of catechol 1,2-dioxygenase.
Plasmid pCL1920 is a low copy vector containing the pSC101 origin
of replication and a gene which confers resistance to
spectinomycin. Digestion of pIB1345 with Sal I and Kpn I yielded a
1.5 kb fragment which was subsequently localized in pCL1920 to
produce pKD8.292 (FIG. 3) in which catechol 1,2-dioxygenase was
expressed from the vector-encoded lac promoter. Transformation of
AB2834/pKD136 with pKD8.243A and pKD8.292 yielded colonies which
were resistant to ampicillin, chloramphenicol, and
spectinomycin.
[0092] Enzyme activities were determined to confirm that E. coli
AB2834/pKD136/pKD8.243A/pKD8.292 was expressing each of the genes
from the ortho cleavage pathway necessary to convert DHS into
cis,cis-muconate. Cultures of AB2834/pKD136/pKD8.243A/pKD8.292 were
grown in LB (1 L) containing IPTG (0.2 mM), ampicillin (0.05 g),
chloramphenicol (0.02 g) and spectinomycin (0.05 g) for 10 h at
37.degree. C., 250 rpm. Cells were harvested and resuspended in 100
mM Tris HCl, pH 7.5, 2.5 mM MgCl.sub.2. After two passages through
a French pressure cell (16,000 psi), the lysate was clarified by
centrifugation (40000 g, 30 min, 4.degree. C.). To measure DHS
dehydratase activity, each assay contained (final volume of 1 mL)
100 mM Tris HCl, pH 7.5, 25 mM MgCl.sub.2, mM DHS, and cellular
lysate. After addition of DHS, formation of protocatechuate
(.epsilon.=3890 M.sup.1 cm.sup.1) was monitored at 290 nm for
several minutes. DHS dehydratase activity measured for three
samples of AB2834/pKD136/pKD8.243A/pKD8-292 was determined to be
0.078 units mg .+-.0.009, where one unit is the amount of enzyme
necessary to convert 1 mmol of DHS to protocatechuic acid in 1
min.
[0093] Catechol 1,2-dioxygenase specific activity was determined
using the same cellular lysate samples produced above. Each assay
contained 100 mM potassium phosphate, pH 7.5, 0.2 mM catechol, and
cellular lysate. Formation of cis,cis-muconate was monitored by
following the increase in absorbance at 260 nm. Assuming a
difference in molar extinction coefficient between cis,cis-muconate
and catechol to be 16,000 M.sup.1 cm.sup.1 under the conditions of
the assay, catechol 1,2-dioxygenase activity in
AB2834/pKD136/pKD8.243A/PKD8-292 was determined to be 0.25 units mg
.+-.0.03, where one unit corresponds to the formation of 1 .mu.mol
of cis,cis-muconate per min.
[0094] To determine the activity of protocatechuate decarboxylase,
AB2834/pKD136/pKD8.243A/pKD8.292 was grown as described previously
in Example 6. Cells were harvested and resuspended in 75 mM
phosphate buffer, pH 7.1. Following disruption by passage through a
French pressure cell (16000 psi), the lysate was clarified by
centrifugation (40000 g, 30 min, 4.degree. C.). Protocatechuate
decarboxylase activity was determined by following the consumption
of protocatechuic acid. Each assay (final volume of 1 mL) contained
75 mM sodium phosphate, pH 6.0, 0.3 mM protocatechuic acid, and
cellular lysate. The loss of absorbance at 290 nm was monitored
over time. Protocatechuate decarboxylase activity in
AB2834/pKD136/pKD8.243A/pKD8.292 was determined to be 0.028 units
mg .+-.0.009, where one unit corresponds to the oxidation of 1 mmol
of protocatechuic acid per min.
Example 7
Conversion of D-Glucose to cis,cis-Muconate
[0095] Microbial synthesis of cis,cis-muconate from D-glucose
utilizing E. coli AB2834/pKD136/pKD8.243A/pKD8.292 proceeded as
follows. One liter of LB medium (in 4 L Erlenmeyer shake flask)
containing IPTG (0.2 mM), ampicillin (0.05 g), chloramphenicol
(0.02 g) and spectinomycin (0.05 g) was inoculated with 10 mL of an
overnight culture of AB2834/pKD136/pKD8.243A/pKD8.292. Cells were
grown at 250 rpm for 10 h at 37.degree. C. The cells were
harvested, resuspended in 1 L of M9 minimal medium containing 56 mM
D-glucose, shikimic acid (0.04 g), IPTG (0.2 mM), ampicillin (0.05
g), chloramphenicol (0.02 g) and spectinomycin (0.05 g). The
cultures were returned to 37.degree. C. incubation. After
resuspension in minimal medium the pH of the culture was closely
monitored, particularly over the initial 12 h. When the culture
reached a pH of 6.5, 5 N NaOH was added to adjust the pH back to
approximately 6.8. Over the 48 h accumulation period, the culture
was not allowed to fall below pH 6.3. After 24 h in minimal medium
12 mM cis,cis-muconate and 1 mM protocatechuate were detected,
using methods described in Example 2, in the culture supernatant
along with 23 mM D-glucose. After 48 h in minimal medium
AB2834/pKD136/pKD8.243A/pKD8.292 had replaced the 56 mM D-glucose
with 17 mM cis,cis-muconate.
Example 7A
Conversion of Glucose to cis,cis-Muconate at 20 L Scale
[0096] FIG. 7A shows the results of a 20 L batch cultivation of
WN1/pWN2.248 for the production of cis,trans-muconic acid. The
culture was induced at OD.sub.600=33 using IPTG (100 mM, 10 mL)
every 6 hours. After about 88 hours, the muconic acid titer was 59
g/L (a 30% yield) and the total amount of muconic acid synthesized
was 1475 g. This corresponds to the conversion of about 1.38 M
glucose to 0.42 M cis,trans-muconic acid in about 88 hours. Table 1
shows the cis,trans-muconic acid production rates at various times
throughout the culture. (Note that the table shows the productivity
post-induction as a function of time. If the outlying data points
are excluded (48 h, after inoculation and 58 h, after inoculation)
the average rate is 1.1 g/L/h.) It was also found that the
recrystallization of IPTG (e.g., in ethyl acetate) can increase the
muconic acid titer. For example, several experiments showed titers
of about 55-60 g/L muconic acid on a 20 L scale, which is about a
17% increase over the about 50 g/L production observed without
recrystallization of IPTG (e.g., a yield of about 30% versus
24%).
TABLE-US-00001 TABLE 1 cis,cis-muconate production rates in the
fermentation. post-induction (h) cis,cis-muconate (g/L) rate
(g/L/h) rate (mmol/L/h) 0 0.52 6 7.46 1.16 8.14 12 16.14 1.45 10.18
18 22.37 1.04 7.31 24 28.26 0.98 6.91 30 35.14 1.15 8.07 36 39.57
0.74 5.20 42 46.72 1.19 8.39 48 47.53 0.14 0.95 53 52.19 0.93 6.56
58 51.55 -0.13 -0.90 66.5 59.22 0.90 6.35
[0097] Following the production of cis,cis-muconate from glucose or
other fermentable carbon source, methods for producing
cis,trans-muconate include (i) providing cis,cis-muconate produced
from a renewable carbon source through biocatalytic conversion;
(ii) isomerizing cis,cis-muconate to cis,trans-muconate under
reaction conditions in which substantially all of the
cis,cis-muconate is isomerized to cis,trans-muconate; and (iii)
separating the cis,trans-muconate and crystallizing the
cis,trans-muconate.
[0098] The isomerization reaction can be catalyzed by an acid, for
example, an inorganic acid. The isomerization reaction can be
carried out in solution at a pH of below pH 7, and preferably at a
pH of about 4 or lower. In some examples, the pH of the
isomerization can be above the value at which one or more of
cis,cis-muconate, cis,trans-muconate, and trans,trans-muconate
precipitates out of solution.
[0099] The isomerization reaction can be carried out at a
temperature greater than room temperature or greater than fermenter
temperature. For example, the isomerization reaction can be carried
out at a temperature of about 30.degree. C. or greater, and
preferably above 60.degree. C. or greater.
[0100] The separating step can include precipitating the
cis,trans-muconate from solution by acidifying the solution.
Preferably, the solution can be acidified to a pH below about 3.
The separating step can include cooling the solution. The solution
can be cooled to a temperature below about 30.degree. C., and
preferably below 0.degree. C.
[0101] Recrystallization can employ an organic solvent. The organic
solvent can include one or more of a polar aprotic solvent (e.g.,
acetic acid, butanol, isopropanol, propanol, ethanol, methanol,
formic acid, water), a polar protic solvent (e.g., dioxane,
tetrahydrofuran, dichloromethane, acetone, acetonitrile,
dimethlyformamide, dimethyl sulfoxide), and a non-polar solvent
(e.g., hexane, benzene, toluene, diethyl either, chloroform, ethyl
acetate).
[0102] In certain embodiments, the method includes removing a salt
from the separated cis,trans-muconate. The salt can include an
inorganic salt.
[0103] In certain embodiments, the method includes isomerizing at
least about 50% of the cis,trans-muconate to trans,trans-muconate,
and preferably more than 95%.
[0104] FIG. 11 shows the results of a 20 L batch cultivation of
WN1/pWN2.248 for the production of cis,trans-muconic acid induced
at OD.sub.600=33 using IPTG (100 mM, 10 mL) every 6 hours. After
about 88 hours, the muconic acid titer was 59 g/L (a 30% yield) and
the total amount of muconic acid synthesized was 1475 g, which
corresponds to the conversion of about 1.38 M glucose to 0.42 M
cis,trans-muconic acid in about 88 hours.
Example 8
In Situ Isomerization of cis,cis-Muconate to cis,trans-Muconate in
a Fermenter
[0105] cis,cis-muconate produced from a renewable carbon source
through biocatalytic conversion (e.g., according to the method of
Examples 7 and 7A) was provided, and the fermentation culture
including the cis,cis-muconate was warmed to 60.degree. C. The
warmed fermentation culture was adjusted to pH 4 by adding 2 N
sulfuric acid over 0.5 h. The acidified culture was allowed to
react for 3.5 h.
[0106] The reaction was monitored by .sup.1H NMR and HPLC equipped
with the Prevail Organic Acid Column (150 mm.times.4.6 mm), to
determine the endpoint of the reaction. These data are presented,
along with control experiments at neutral pH, in FIGS. 7-10. In
general, such isomerization reactions can be monitored to determine
appropriate reaction parameters (e.g., time, temperature, pH, and
the like).
[0107] FIG. 5 shows .sup.1H NMR traces for a cis,cis- to
cis,trans-muconate isomerization reaction at pH 7 in a crude
fermentation broth. The time traces from 0 to 1.25 and 3.25 hours
demonstrate that there is essentially no isomerization from cis,cis
to cis,trans muconic acid at neutral pH (e.g., which is the
approximate pH level during an actual fermentation). Thus, no or
negligible isomerization of cis,cis-muconate occurs during an
actual fermentation.
[0108] FIG. 6 shows .sup.1H NMR traces for a muconate isomerization
reaction at pH 4 in a crude fermentation broth. The time traces
from 0 to 1.25 and 3.25 hours demonstrate that isomerization from
cis,cis to cis,trans-muconic acid proceeds rapidly at acidic pH,
and that the isomerization is essentially complete after about 1.25
hours.
[0109] FIG. 7 shows HPLC traces for a muconate isomerization
reaction at pH 7. FIG. 9 shows a time course for a muconate
isomerization reaction at pH 7. As with the .sup.1H NMR traces,
these HPLC traces and time course demonstrate that there is
essentially no isomerization from cis,cis to cis,trans-muconic acid
at neutral pH.
[0110] FIG. 8 shows HPLC traces for a muconate isomerization
reaction at pH 4. FIG. 10 shows a time course for a muconate
isomerization reaction at pH 4. As with the .sup.1H NMR traces,
these HPLC traces and time course demonstrate that that
isomerization from cis,cis- to cis,trans-muconic acid proceeds
rapidly at acidic pH, and that the isomerization is essentially
complete after about 1.25 hours.
Example 9
Separation of cis,trans-Muconic Acid from Fermentation Broth by
Acidification, Precipitation, and Filtration
[0111] Following isomerization (e.g., as in Example 8), broth
containing cis,trans-muconic acid was cooled to approximately
ambient temperature and the cells, cell debris and precipitated
solids were removed from the culture broth by centrifugation.
Alternatively, such solids can be removed by filtration (e.g.,
through a 100 kD SARTOCON.RTM. Slice cassette). The cell-free broth
was then clarified by filtration (e.g., through a 10 kD
SARTOCON.RTM. Slice cassette), to remove proteins.
[0112] After filtration, the pH of the clarified broth was adjusted
to pH 1.5 by adding concentrated sulfuric acid. The amount of
cis,trans-muconate that precipitates at various pH values is shown
in Table 2.
TABLE-US-00002 TABLE 2 The precipitation of cis,trans-muconate at
different pH values. ctMA in precipitate % ctMA in filtrate
ctMA/total MA pH (weight %) (weight %) (HPLC %) 4.7 0 100 98 4.0 22
75 94 3.5 58 26 86 3.0 61 22 83 2.5 64 18 87 2.0 18 86
[0113] The acidified broth was chilled to 4.degree. C. for 1.5 h
without agitation, during which time crude cis,trans-muconic acid
precipitated as a slightly yellow solid. This material was
recovered by filtration and comprised about 60% of the
cis,trans-muconic acid present in the clarified broth. The
precipitation can be allowed to continue for a longer period of
time (e.g., overnight) and/or at a lower temperature (e.g.,
-20.degree. C.), to increase product recovery while mitigating salt
contamination.
[0114] The filtrate contained further cis,trans-muconic acid. In
order to recover the further cis,trans-muconic acid, the filtrate
was evaporated under reduced pressure, to reduce the volume by
about 50%. The concentrated filtrate was chilled to -20.degree. C.
overnight, during which time a second crop of crude
cis,trans-muconic acid precipitated. The precipitate was again
recovered by filtration.
[0115] The crude cis,trans-muconic acid solids were combined and
crystallized using acetonitrile to produce purified
cis,trans-muconate as a white solid. Crystallization using methanol
provided similar yields. Methanol also mitigated salt contamination
in the purified product.
[0116] FIG. 4A shows an .sup.1H NMR spectrum of crystallized
cis,trans-muconic acid. FIG. 4B shows an .sup.1H NMR spectrum of
crystallized cis,trans-muconic, resuspended in a minimal salts
medium lacking glucose. The FIG. 4B spectrum approximates the NMR
spectral shifts in cis,trans-muconic acid caused by other
components in the fermentation broth, and thus allows for
comparisons monitoring the cis,cis to cis,trans isomerization
reaction in the fermentation broth.
Example 10
Extraction of cis,trans-Muconic Acid from Fermentation Broth Using
an Organic Solvent
[0117] Advantageously, cis,trans-muconic acid is surprisingly and
unexpectedly more soluble in organic solvents than either of the
cis,cis or trans,trans isomers. Therefore, a separating step (e.g.,
the separating step of Example 8) can include extracting the
cis,trans-muconate from solution (e.g., a fermentation broth) using
an organic solvent.
[0118] The solution from which the cis,trans-muconate is extracted
can be a whole culture fermentation broth or a cell free, protein
free fermentation broth. Cells can be removed from a broth, for
example, by filtration (e.g., passing the broth through a 0.1 .mu.M
hollow fiber filtration unit). Proteins can be removed from a
broth, for example, by filtration (e.g., through a 10 kD tangential
flow filtration system available, for example, from
SARTOCON.RTM.).
[0119] The organic solvent for extraction (e.g., solvent that is
immiscible with an aqueous phase) can include, for example, one or
more of: methyl isobutylketone (MIBK), ethyl acetate, isopropyl
acetate (propyl acetate), heptanes (mixture), methyl
tert-butylether, xylenes, methylene chloride, cyclohexanol,
decalin, tetralin, tetralone, cyclohexane, butyl acetate, methyl
tetrahydrofuran (THF), cyclohexanone/cyclohexanol (commercial
mixture), 1-octanol, isoamyl alcohol, and 2-ethylhexanol.
[0120] Other organic solvents which can be added to the aqueous
phase and which will facilitate both the extraction and concurrent
or subsequent esterification of the cis,trans-muconate can include,
for example, one or more of: methanol, ethanol, propanol,
isopropanol, acetic acid, acetonitrile, and acetone, as well as
butanols such as 1-butanol and isobutanol, and other alcohols which
are not completely miscible with water.
[0121] The solvent extraction can be carried out at a pH of below
about 4, e.g., at a pH where the cis,trans-muconic acid is
sufficiently protonated that it will partition into the organic
solvent used for the extraction. Even at pH levels low enough to
induce precipitation, a fraction of the protonated
cis,trans-muconic acid can remain in solution. For example, and as
shown in Table 2 in Example 9 above, at pH 3 about 60% of the
cis,trans-muconic acid originally in the solution precipitates and
can be separated by filtration. However, the approximately 40% of
the cis,trans-muconic acid remaining in solution cannot be
separated by filtration but can be recovered by extraction.
Accordingly, solvent extraction can increase the isolation yield of
cis,trans-muconate relative to a method that does not include
extracting the cis,trans-muconic acid from solution using an
organic solvent. It is also clear from the data in Table 2 of
Example 9 that the extraction of cis,trans-muconate can proceed
even if some portion of the cis,trans-muconate has precipitated and
is not in the aqueous solution.
[0122] The solvent extraction can also include separating the
cis,trans-muconate from an inorganic salt (e.g., ammonium sulfate,
calcium sulfate). Furthermore, solvent extraction can produce a
more pure cis,trans-muconate than precipitation which can also
cause the precipitation of, and therefore contamination with,
inorganic salts.
[0123] Selection of solvent and/or pH parameters can be facilitated
by a series of simple measurements. For each potential solvent,
extractions can be performed (e.g., to measure partition
coefficients) on all three muconic acid isomers (cis,cis-,
cis,trans-, and trans,trans-isomers). Each isomer can also be
tested against a range of pH values using increments (e.g., 0.5)
between about pH 1 and below pH 7.
Example 11
Separation of cis,trans-Muconic Acid from Fermentation Broth by
Solvent Extraction
[0124] Fermentation broth was obtained that had been isomerized to
cis,trans-muconic acid, and was acidified to a pH of about 3. The
solid cis,trans-muconic acid was removed by filtration to leave
acidified fermentation broth containing approximately 5 to 10 grams
per Liter of cis,trans-muconic acid.
[0125] To individual 50 mL conical centrifuge tubes each containing
15 mL of the filtered broth were added 15 mL of each solvent listed
in tables below. Each tube was agitated for two minutes and the
organic and aqueous phases allowed to separate. The aqueous layer
was separated from the solvent layer by pipette and placed into a
new 50 mL conical tube. Both the aqueous and solvent phases of each
extraction were analyzed for muconic acid using HPLC. A second
extraction was performed on each of the separated aqueous layers
using fresh solvent. Again the samples were agitated, allowed to
settle and the aqueous and organic phases were separated and
analyzed.
[0126] The results are shown in Tables 3, 4, and 5 below. Different
broth samples with different amounts of cis,trans-muconic acid were
used to generate the results in each table.
TABLE-US-00003 TABLE 3 Broth containing 9.82 g/L cis,trans-muconic
acid. Muconic acid Muconic acid Extraction in organic in aqueous
Partition Solvent number phase phase coefficient None 9.82 tBME 1
6.53 2.05 3.19 tBME 2 1.23 0.72 1.71 Octanol 1 5.40 2.19 2.47
Octanol 2 0.53 1.52 0.35 1-butanol 1 6.26 1.10 5.69 1-butanol 2
0.51 0.27 1.89
TABLE-US-00004 TABLE 4 Broth containing 10.69 g/L cis,trans-muconic
acid. Muconic acid Muconic acid Extraction in organic in aqueous
Partition Solvent number phase phase coefficient none 10.69
1-pentanol 1 8.29 1.29 6.43 1-pentanol 2 1.06 0.30 3.53 cyclohexane
1 0.00 10.47 0.00 cyclohexane 2 0.00 10.20 0.00 n-butyl acetate 1
6.78 3.96 1.71 n-butyl acetate 2 3.55 2.02 1.76 MEK 1 9.65 2.63
3.67 MEK 2 1.99 0.84 2.37 MeTHF 1 7.40 0.65 11.38 MeTHF 2 0.51 0.00
large cylcohexanol 1 8.62 0.98 8.80 cylcohexanol 2 1.05 0.28 3.75
decalin 1 0.00 10.25 0.00 decalin 2 0.00 12.99 0.00
TABLE-US-00005 TABLE 5 Broth containing 4.86 g/L cis,trans-muconic
acid. Muconic acid Muconic acid Extraction in organic in aqueous
Partition Solvent number phase phase coefficient none 4.86
cyclohexanone 6.11 0.21 29.10 cyclohexanone 0.00 0.23 large xylene
0.00 5.15 0.00 xylene 0.00 4.82 0.00 isoalcohol 4.87 0.63 7.73
isoalcohol 0.49 0.04 12.25 ethyl hexanol 3.83 0.89 4.30 ethyl
hexanol 0.79 0.20 3.95
[0127] To test the ability of solvent extraction to recover
cis,trans-muconic acid after acidification to sufficiently low
levels that significant amounts of the cis,trans-muconic acid has
precipitated, a solution of cis,trans-muconic acid in a solution of
M9 salts was used to simulate a fermentation broth. 15 grams of
cis,trans-muconic acid were added to 250 mL of M9 salts giving an
approximate titer of 60 g/L. This was achieved by raising the pH to
7.0 using sodium hydroxide. The broth was then acidified with
sulfuric acid to pH 3.0 causing the cis,trans-muconic acid to
precipitate. The solid precipitate was left in the acidified
mixture, and the entire slurry was extracted twice with solvent as
described above. The results are shown in Table 6.
TABLE-US-00006 TABLE 6 Broth containing 63.34 g/L cis,trans-muconic
acid, including precipitated muconic acid. Muconic acid Muconic
acid Extraction in organic in aqueous Partition Solvent number
phase phase coefficient none 63.34 cyclohexanone 1 51.05 2.92 17.48
cylcohexanone 2 2.10 0.20 10.50 MeTHF 1 47.10 2.03 23.20 MeTHF 2
1.64 0.11 14.91 cyclohexanol 1 51.37 10.53 4.88 cyclohexanol 2 3.75
2.71 1.38 tBME 1 0.00 54.00 0.00 tBME 2 0.00 59.88 0.00
Example 12
Isomerization of cis,trans-Muconic Acid to trans,trans-Muconic Acid
Catalyzed by Iodine
[0128] A mixture containing cis,trans-muconic acid (1.00 g), a
catalytic amount of I.sub.2 (53 mg), and MeCN (35 ml) was heated to
reflux for 11 h. After cooling to room temperature, the
precipitated solid was filtered off and washed with cold MeCN.
After drying under vacuum, 0.80 g (80% yield) of purified
trans,trans-muconic acid was present as a tan-colored powder. The
material obtained by this procedure was confirmed to be
trans,trans-muconic acid by .sup.1H and .sup.13C NMR spectroscopy.
The isomerization reaction proceeded better in nonpolar solvents
(e.g., THF) than in a number of other tested solvents.
Example 13
Isomerization of cis,trans-Muconic Acid to trans,trans-Muconic Acid
Catalyzed by a Hydrogenation Catalyst
[0129] A mixture containing cis,trans-muconic acid (1.00 g) and a
catalytic amount of palladium supported on carbon (Pd/C, 5%) is
prepared in 50 mL of methanol. The methanol reaction mixture is
brought to reflux for 1 hour, cooled to room temperature, and then
the supported palladium catalyst is removed by filtration. The
remaining reaction solution is evaporated to about one-half the
original volume, then diluted with one volume of MeCN. Evaporation
under reduced pressure is continued until the methanol is removed
and the trans,trans-muconic acid begins to fall out of solution.
The resulting solid is filtered off and washed with cold MeCN.
After drying under vacuum, about 0.80 g (80% yield) of purified
trans,trans-muconic acid can be present as a tan-colored powder.
The material obtained by this procedure can be confirmed to be
trans,trans-muconic acid by .sup.1H and .sup.13C NMR
spectroscopy.
Example 14
Isomerization of cis,cis-Muconic Acid to trans,trans-Muconic Acid
Catalyzed by a Hydrogenation Catalyst
[0130] A mixture containing cis,cis-muconic acid (1.00 g) and a
catalytic amount of palladium supported on carbon (Pd/C, 5%) is
prepared in 50 mL of methanol. The methanol reaction mixture is
brought to reflux for 1 hour, cooled to room temperature, and then
the supported palladium catalyst is removed by filtration. The
remaining reaction solution is evaporated to about one-half the
original volume, then diluted with one volume of MeCN. Evaporation
under reduced pressure is continued until the methanol is removed
and the trans,trans-muconic acid begins to fall out of solution.
The resulting solid is filtered off and washed with cold MeCN.
After drying under vacuum, about 0.80 g (80% yield) of purified
trans,trans-muconic acid can be present as a tan-colored powder.
The material obtained by this procedure can be confirmed to be
trans,trans-muconic acid by .sup.1H and .sup.13C NMR
spectroscopy.
[0131] While the invention has been particularly shown and
described with reference to specific embodiments, it should be
understood by those skilled in the art that various changes in form
and detail can be made without departing from the spirit and scope
of the invention as defined by the appended claims.
* * * * *